CD8+ T-cell T-cells can be a central role in immune control of HIV infection. However, the CD8+ T-cell responses that should be induced by an HIV vaccine in order to be optimally effective are yet to be established. Work we have done in the course of the previous 5 years of this grant have focused on the southern African HIV epidemic, the region worst affected by HIV and most urgently in need of an effective vaccine. One major conclusion that can be drawn from the work we have done in the past 5 years of this grant is that immune control of HIV infection is facilitated by a broad Gag-specific CD8+ T-cell response, in particular by responses in Gag, but also to a lesser extent in Pol, that can drive selection pressure on the virus. It is apparent that some of the most efficacious responses are subdominant to other, ineffective responses. Epitope efficacy differs between proteins (Gag>Pol>Acc-Reg>Env), but also within proteins. Epitope efficacy also differs acording to HLA restriction. HLA-B-restricted responses have the greatest impact on viral setpoint, and drive the strongest selection pressure on the virus. HLA-C-restricted responses are associated with high viral setpoint even when they are Gag-specific. The goal of the first specific aim of these proposed studies is, using a novel approach to achieve rapid definition of CTL epitopes, to identify those responses, including subdominant responses, in Gag and Pol that are most effective in inhibition of HIV replication. It is quite clear that current knowledge of the optimal epitopes targeted, and the CD8+ T-cell response hierarchy even for the most prevalent HLA alleles, remains inadequate in order to understand the CD8+ T-cell response in natural HIV infection and what perturbations of this response hierarchy may be needed to optimise the CD8+ T-cell contribution to immune control of HIV. For an allele such as A*6802, present in 1/6 of the southern African population, currently described optimal epitopes account for only 1% of the actual responses made by A*6802-positive subjects that are restricted by that alelle. The novel approach, involved use of peptide-MHC tetramers, is rapid and inexpensive, has been validated by us as shown in preliminary data presented, and crucially provides the further opportunity to assess the efficacy of purified antigenic-specific cells in viral inhibition assays. The second aim of these studies is to investigate the ability of a T-cell vaccine in humans to alter the response hierarchy observed in natural infection. We will study the South African subjects who were vaccinated with the MRK Ad5 vaccine (HVTN 503, 'Phambili' study), which expressed Gag, Pol and Nef. Using our database of 1,015 south African study subjects in whom we have characterized the CD8+ T-cell responses against C clade infection, using a panel of 410 overlapping 18mer peptides, we will be able to determine whether this MRK Ad5 T-cell vaccine, which has already been shown to boost specifically Gag, Pol and Nef responses in post-infection vaccinees, can alter the response hierarchy that is seen in non-vaccinated infected people. This would be an important prerequisite for a T-cell vaccine to be effective. The final specific aim of these proposed studies is to investigate the impact of viral adaptation to the CD8+ T-cell response hierarchy. We have shown in the previous 5 years' work that viral adaptation is occurring at a population level, such that escape mutants in even the most protective epitopes are accumulating over time. How fast, and the extent to which, these escape mutants accumulate will be considered in these proposed studioes, to determine whether viral adaptation in fact is likely to have an impact on current T-cell vaccine design.